Many academic and industrial processes rely on in vitro culturing to generate greater amounts of cells of interest. In these processes, a sample of cells is generally placed in a vessel, provided with nutrients, and agitated. After a sufficient period of time to allow production and growth of new cells, the cultured cells are removed from the vessel and purified. For example, small-scale cell culturing has traditionally been performed in shallow shaker flasks. The cells and nutrient media are combined in the flask and a mechanical or magnetic stirring mechanism circulates the mixture. This circulation is important to prevent the cells from settling to the bottom of the culture vessel due to gravity and to ensure sufficient nutrient transfer to and waste bioproducts from the growing cells.
These mass transfer requirements are important for all forms of culturing cells, for example, in the form of a single cells, multicellular aggregates or cells attached to a substrate. In many culture systems, particularly mammalian culture systems, the formation of multicellular aggregates is important to accumulate large biomasses. Mammalian cells are also often cultured in an attached state to mimic the in vivo environment. When shear forces become too high, these forces can deform and damage individual cells, impede cell growth, prevent or limit the aggregation of cells, or pull apart aggregates and tear the walls of adjacent cells.
Several research groups have studied the effects of shear stress on cell cultures. Stathopoulos et al., Biotechnol. Bioeng. 27:1021-1026 (1985), studied shear stress effects on human embryonic kidney cells in vitro and reported that forces of 0.65 N/m.sup.2 had significant effects on cell morphology and forces higher than 2.6 N/M.sup.2 caused marked reduction in cell viability. Croughan et al., Biotechnol. Bioeng., 29:130-141 (1987), and Croughan et al., Biotechnol Bioeng. 33:731-744 (1989), evaluated growth of FS-4 (human diploid fibroblasts) microcarrier cultures at various impeller rotation rates in spinner vessels equipped with magnetic stir bars. Croughan et al. (1987) suggests that this analysis would not apply to vessels having different geometries.
A variety of geometries have been proposed for cell culture vessels. Tsao et al., "Fluid Dynamics Within a Rotating Bioreactor in Space and Earth Environments," Journal of Spacecraft and Rockets 31:937-943 (1994), and Tsao et al., "Mass Transfer Characteristics of NASA Bioreactors by Numerical Simulation," Advances in Heat and Mass Transfer in Biotechnology (Clegg, S., ed.) HTD-Vol. 355:69-73 (1997), disclose a bioreactor having a culturing vessel with two concentric, independently rotating cylinders. U.S. Pat. Nos. 5,026,650 (Schwartz., issued Jun. 25, 1991); 5,155,034 (Wolf et al., issued Oct. 13, 1992); and 5,153,133 (Schwartz et al., issued Oct. 6, 1992) disclose a bioreactor having a horizontally disposed cylindrical culture vessel that rotates about its longitudinal axis. The vessel contains a coaxially-disposed, oxygen-permeable membrane and access ports to inject or withdraw nutrient media.
U.S. Pat. No. 5,151,368 (Brimhall et al., issued Sep. 29, 1992) describes a dual-axis continuous flow bioreactor for handling high solids loaded materials such as coal and mineral ores. The bioreactor is mounted to a horizontal axle, corresponding to the bioreactor's longitudinal axis, and moves in a circular path about its vertical shaft while simultaneously rotating about its horizontal axis by an interlocking set of bevel ring gears. The vessel contains several conduits for introducing and withdrawing media and gases.
U.S. Pat. No. 5,565,361 (Mutsakis et al., issued Oct. 15, 1996) describes a bioreactor containing a porous, fibrous sheet material that acts as a motionless mixing element and as a substrate for attaching the cultivating cells. U.S. Pat. No. 5,622,857 (Goffe, issued Apr. 22, 1997) describes a bundled hollow fiber bioreactor and its use to prepare eucaryotic cells. The flow of oxygenated nutrient medium is maintained at a sufficient pressure to prevent the formation of bubbles that may block flow of media through the fibers. U.S. Pat. No. 5,688,687 (Palsson et al., issued Nov. 18, 1997) describes a bioreactor for mammalian cell growth having a circular cell growth chamber between a planar cell bed and a gas permeable membrane. Media inlet and outlets are arranged in a concentric circular arrangement and provide for the delivery of nutrients to the cells. U.S. Pat. No. 5,605,835 (Hu et al., issued Feb. 25, 1997) describes a bioreactor that immobilizes animal cells in an insoluble, biocompatible matrix. Selective membranes allow for the separation of cell nutrients and cell wastes while collecting the desired cell products within the cell chamber.
There is a need for improved bioreactors and cell culturing methods that enhance the production and growth of cells without creating shear forces that damage cultivating cells or multicellular aggregates.